1. Field of the Invention
This invention relates generally to the field of cold storage preservation of living cells, and more particularly to the process for preparing such cells for deep-freezing preservation, and the subsequent process of thawing to revive such cells.
2. Description of the Prior Art
The use of cold temperatures for the preservation of biological materials is well known. The field of artificial insemination of animals is dependent upon the ability to freeze a specimen of animal semen for storage and shipment, to thaw it for use, and to have a sufficient number of sperm cells survive to be effective. The freezing and thawing processes are a severe shock to any living cell and can cause serious chemical and structural phase changes in the cell.
Glycerol was found, in 1949, to provide a protective effect for bovine semen against the shock of deep freezing -- down to liquid nitrogen temperatures. This discovery was extended to the preservation of a variety of biological materials, including whole blood. The glycerol normally is used in a concentration of 5% to 10% of the semen or other biological material.
Unfortunately, glycerol has been found to be toxic at the concentrations in use, and particularly so when the specimen is exposed to ambient temperatures for extended periods of time. This adverse reaction has precluded its use with some biological materials. For some materials, including whole blood, a low temperature dialysis procedure is required for removal of the glycerol after the material is thawed and prior to its use.
A number of cryoprotective diluents, other than glycerol, have been tried and reported in the patent and technical literature. These include: dimethylsulfoxide, high molecular weight sugars (e.g. lactose, raffinose, etc.) and polymeric hydrocarbons such as polyvinylprolidone. Organic buffering afents such as TRIS (trishydroxymethylamino methane) and TES N tris(hydroxymethyl) methyl-2-aminoethane sulfonic acid have also been incorporated in some cryoprotective diluents. Examples of other diluters are taught in the patents to Smith et al., U.S. Pat. No. 3,185,623; and to Folkers et al. U.S. Pat. No. 3,306,818. However, glycerol has remained the cryoprotective diluent of choice for commercial processing of "frozen semen."
The freezing process per se, and particularly the rate of cooling for preparing "frozen semen," has also been a subject of extensive study. The general consensus from these studies recommends, for 1 ml. ampules of semen, a slow cooling rate (1.degree.C per minute) from approximately + 4.degree.C to -15.degree.C followed by a nominal 3-fold increase in the cooling rate. For "ampule freezing," this continues to be the generally used procedure.
An alternative "vapor freezing" technique, particularly for freezing semen in glass or plastic straws has also been employed. This involves suspending the straws filled with semen and diluent above the surface of a liquid nitrogen bath. After the samples are frozen, they are then immersed in the liquid nitrogen for storage. This technique is still widely used for both ampules and straws.
A third technique involves "pellet freezing" of semen and diluent. A droplet of approximately 0.07 ml. diluted semen is placed directly upon the surface of "dry ice" and allowed to remain until frozen. The pellet is subsequently transferred into liquid nitrogen for storage.
All of the above described methods have been predicated upon empirically derived slow cooling rates, usually involving the classical "supercooling temperature curve." This classical curve denotes crystallization from a supercooled state. The "pellet freezing" technique may appear to be an exception, but, in fact, is not. The sublimation of CO.sub.2 increases in the region immediately below the droplet of semen, thus forming essentially an insulating blanket of gas which effectively slows the cooling rate. As the temperature of the semen droplet decreases, the CO.sub.2 evolution slows, and the cooling rate of the droplet is then increased. As a consequence of these interactions between the semen droplet and the "dry ice" surface, the relative cooling rate of the sample is still that which is generally characterized as slow freezing.
The rate of thawing of semen samples has also received some study, but relatively little in comparison to the freezing process. Generally, glycerol protected samples thawed at body temperature or in ice water have given acceptable fertility rates.